Abstract

This is the first report enumerating a superb antiproliferative effect
of both sulindac and exisulind on hepatocellular cancer cell lines. The
growth inhibition and cytotoxicity of sulindac in human hepatocellular
carcinoma cell lines HepG2, Huh-7, and KYN-2 were investigated by
studying cell growth, cell cycle distribution, and induction of
apoptosis. In the presence of sulindac, there was a marked time- and
dose-dependent decrease in cell proliferation and viability. Also,
exisulind exhibited a similar growth-inhibitory effect on the KYN-2
cell line. The findings of this study suggest that sulindac exhibits a
growth-inhibitory effect on human hepatocellular carcinoma cell lines;
therefore, these drugs might serve as an effective tool for
hepatocellular carcinoma chemoprevention.

Introduction

Accumulating evidence indicates that the
NSAIDs
2
possess an antiproliferative effect and induce apoptosis and tumor
regression
(1, 2)
. Initially, the research regarding the
chemopreventive effect of NSAIDs was largely confined in
colorectal-originated tumor cell lines; however, rapid development in
this field urged researchers to extend this finding to other
carcinomas, including breast, prostate, and pancreatic carcinoma cell
lines
(2,
3,
4)
. HCC is one of the most lethal malignancies
and ranks worldwide as the seventh most common cancer
(5)
.
Both colorectal cancer and HCC have a unique similarity in their
natural history of development, often characterized by multistage tumor
development with distinct morphological and biological phases.
Extensive work regarding the chemopreventive effect of a new generation
of NSAIDs, sulindac, in colorectal lesions and cell lines has been
accomplished
(6, 7)
; however, thus far, there has been no
report in HCC. Hence, the present study was undertaken to analyze the
effect of sulindac and its irreversible oxidized derivative, sulindac
sulfone (exisulind), in three human HCC cell lines. Also, the rationale
for elucidation of the effect of sulindac in HCC cell lines was
proposed by a report indicating that well-differentiated HCCs
express COX-2 more frequently and strongly than less-differentiated
ones
(8)
.

Materials and Methods

Cell Lines and Cell Culture.

Three HCC-derived cell lines designated KYN-2, HepG2, and Huh-7 were
used. The KYN-2 cell line was kindly provided by the First Department
of Pathology, Kurume University School of Medicine (Kurume,
Japan). HepG2 and Huh-7 cells were obtained from the American
Type Culture Collection (Manassas, VA). Cells were grown in DMEM
supplemented with 5% fetal bovine serum (Life Technologies,
Gaithersburg, MD), penicillin (100 units/ml), and streptomycin (100μ
g/ml) in an atmosphere of 5% CO2.

Immunostaining and RT-PCR for COX-2 Expression.

Expression of COX-2 was checked at the protein and mRNA levels by
immunohistochemistry and RT-PCR, respectively, in all three cell lines
used for this study. Immunohistochemistry was done by the
avidin-biotin-peroxidase complex method as described elsewhere
(4)
. Briefly, cell smears were fixed in 4%
paraformaldehyde, endogenous peroxidase activity was quenched with 3%
H2O2, and nonspecific
binding was blocked by normal rabbit serum. Slides were treated with
mouse anti-human COX-2 antibody (Cayman Chemical, Ann Arbor, MI) at a
1:500 dilution overnight at 4°C. All other steps were done using a
Histofine SAB-PO(R) (Nichirei Corp., Tokyo, Japan) kit according to the
manufacturer’s instructions. The reaction product was visualized by
3-amino-9-ethylcarbazole (Histofine, Tokyo, Japan).
Counterstaining was done using hematoxylin.

For RT-PCR, total RNA was extracted using the Tri reagent (Molecular
Research Center, Inc., Cincinnati, OH) following the manufacturer’s
instructions. RT-PCR was performed as described previously
(4)
.

Growth Inhibition and MTT Assays.

Approximately 5 × 104 cells were
plated in 60-mm-diameter Petri dishes in triplicate. Cells were allowed
to grow, and after 24 h sulindac (Sigma Chemical Co., St. Louis,
MO) and exisulind (a generous gift from Merck, Rahway, NJ) at various
concentrations (25, 50, and 100 μm) dissolved in DMSO
were added to the treatment dishes. The final concentration of DMSO was<0.1%. Cells were harvested at definite time intervals by
trypsinization, and aliquots were counted using a hematocytometer. On
day 3, only KYN-2 cells were harvested by trypsinization and were used
for MTT assay (Boehringer Mannheim, Mannheim, Germany). This assay
relies on the ability of viable cells to reduce the tetrazolium salt
MTT metabolically to a purple formazan product, which can be quantified
colorimetrically. One hundred-microliter aliquots of cells were
transferred to triplicate wells of a flat-bottomed 96-well microtiter
plate and treated with 10 μl of MTT for 4 h at 37°C. After
that, 100 μl of solubilization solution were added, and the mixture
was incubated at 37°C for overnight. The solubilized formazan product
was spectrophotometrically quantified using a microtiter plate reader
(EAR 400; FW Slt-Labinstruments, Groedig, Austria) at 550-nm
wavelength. The morphology of the KYN-2 cells both in the sulindac and
control group was checkd by a phase-contrast light microscope.

Cell Cycle Analysis.

The proportion of cells in
G0-G1, S, and
G2-M was determined by flow cytometric analysis
of DNA content (EPICS Elite ESP flow cytometer; Coulter Electronics,
Miami, FL). Cell cycle distribution in KYN2 cells was measured after
24 h of treatment with 100 μm sulindac. In brief,
cell suspension was prepared by trypsinization, and ∼2 × 106 cells/ml were washed twice with PBS.
The cells were resuspended with 10 ml of 70% ethanol (−20°C),
incubated at 4°C for 4 h, washed twice in PBS, incubated
with RNase (Sigma) at a concentration of 0.25 mg/ml at 37°C for 15
min, followed by treatment with propidium iodide (50 μg/ml), and
incubated for 30 min at 4°C in the dark. DNA histograms were analyzed
using Multicycle AV software (Phoenix, San Diego, CA) to evaluate cell
cycle compartments.

Detection of Apoptosis.

Apoptosis was detected by an annexin V-FITC kit (Immunotech, Marseille,
France) according to the manufacturer’s instructions. Briefly,
KYN-2 cells were collected by trypsinization after a 72-h treatment,
and the number of cells was adjusted to ∼1 × 106 cells/ml Cells were washed with ice-cold DMEM
and were centrifuged to collect the cell pellet. Cell pellet was
resuspended in ice-cold binding buffer. After that, annexin V-FITC (10μ
l/ml) and PI (10 μl/ml) solutions were added to the cell
suspension and mixed gently. The tube was then incubated for 10 min in
the dark before being analyzed by the flow cytometer. All steps were
carried out on ice. Aliquots of stained cells were smeared onto glass
slides, and morphological changes were examined under a fluorescence
microscope.

Statistical Analysis.

A minimum of three experiments measuring the growth inhibition was
performed, and the mean of the inhibitory effect was expressed as
percent inhibition calculated as (number of treated cells/number of
control cells × 100. Pairwise group comparisons between
different groups were done using the unpaired Student’s t
test. Significant differences were assumed when the chance of
differences arising from a sampling error was <1 in 20
(i.e.,P < 0.05).

Results

Growth Inhibition.

All three cell lines used in this study, KYN-2, HepG2, and Huh-7,
expressed COX-2 strongly, and also the expression of COX-2 at the mRNA
level was confirmed by RT-PCR (Fig. 1)
⇓
. Sulindac exhibited a statistically significant dose-dependent
growth-inhibitory effect on all HCC-derived cell lines evaluated in
this study (Fig. 2a)
⇓
. Supplementation of KYN-2 cell culture medium for 72 h with 25, 50, and 100 μm sulindac produced
almost 10, 48, and 70% reduced cell growth, respectively, when
compared with cells in medium supplemented with DMSO only (Fig. 2b)
⇓
. As shown in Fig. 2b⇓
, at comparable
concentrations, exisulind had a comparatively greater dose-dependent
inhibitory effect than that of the sulindac at 72 h of treatment;
however, there was no statistical difference between the two groups.
Moreover, the growth-inhibitory effect of sulindac on KYN-2 cells was
found to be time dependent, because the inhibitory effect became
gradually stronger with the passage of time after treatment, with the
most significant effect observed at 72 h. (Fig. 2c)
⇓
. As
shown in Fig. 2d⇓
, the results of manual counting of cell
numbers conformed well with those performed with the MTT assay.
Sulindac- and exisulind-induced morphological changes were evident by
72 h of treatment in all cell lines. Cells in dishes supplemented
with sulindac or exisulind became sparse, rounded, and detached from
the dishes (Fig. 2f)
⇓
.

Growth-inhibitory effect of sulindac and sulindac sulfone.
A concentration-dependent growth inhibitory effect of sulindac on all
HCC cell lines was noticed (a), and a similar effect was
also noted for sulindac sulfone in KYN-2 cells (b). A
time-dependent effect of sulindac on KYN-2 cells was evident at a 100μ
m dose (c). The manually counted data of
cell number were reproducible by the MTT assay (d).
e and f, Morphological characteristics of
KYN-2 cells before (e) and after (f)
sulindac treatment.

Cell Cycle Effect.

DNA histograms prepared from cells cultured in medium supplemented with
sulindac showed accumulation of cells in
G0-G1 and a corresponding
reduction in percentages of cells in S and G2–M
(Fig. 3)
⇓
. In contrast to the apoptotic effect, arrest of the cell cycle was not
time dependent. Sulindac produced cell quiescence during the first
24 h of cell culture in the preconfluent state, and after that the
cell cycle progressed to S and G2-M as noted in
the control cells at 48 h after sulindac treatment (data not
shown).

Discussion

The potential for sulindac as an antineoplastic agent has been
unanimously established in colorectal carcinoma cell lines.
Subsequently, similar results have been reported in several other
carcinomas
(2,
3,
4)
; however, the effect has yet to be
elucidated in HCC-originated cell lines. Sulindac, a new generation of
NSAID, inhibits both isoforms of COX (COX-1 and COX-2), key enzymes
that catalyze the formation of prostaglandins from arachidonic acid.
This inhibitory effect of sulindac is attributable to its
antineoplastic effect, although much controversy remains around this
plausible hypothesis. Much of this controversy looms around a similar
antineoplastic effect exerted by exisulind, which has virtually no
inhibitory effect on either of the COX enzymes
(3)
. As
established previously in colorectal carcinoma cell lines, similarly
both sulindac and exisulind had a uniform growth-inhibitory effect on
all HCC-originated cell lines used for this study.

All of the three cell lines used in this study had different
differentiation statuses, with HepG2 and Huh-7 being well
differentiated
(9)
and KYN-2 being less differentiated
(10)
. Koga et al.(8)
reported
that COX-2 is involved in early stages of HCC carcinogenesis and is
expressed frequently in well-differentiated carcinomas. However, in
this study, irrespective of their differentiation status, all three
cell lines expressed COX-2 both at the protein and mRNA levels.
Supplementation of culture medium with both sulindac and its
irreversible oxidative form exisulind at comparable concentrations
produced a concentration-dependent growth-inhibitory effect. A growing
body of evidence suggests that exisulind induces apoptosis in various
tumor cell lines and inhibits chemically induced colonic
(11)
and mammary carcinogenesis
(2)
. In these
reports the authors concluded, and we concur with them, that the
antineoplastic effect of sulindac may be irrespective of its inhibitory
effect on COX enzymes and prostaglandin biosynthesis. It is worth
mentioning that a comparable growth-inhibitory effect of sulindac could
be achievable at a much lower concentration (100μ
m) in HCC cell lines than in other cell lines,
because sulindac had a significant inhibitory effect at 100μ
m in HCC cell lines, whereas in pancreatic and
colon cancer cell lines it was 200 and 400 μm,
respectively
(4, 7)
. Sulindac is a prodrug and is
metabolized to sulindac sulfide by the gut flora and in the liver
before it exerts its effect on COX enzymes
(12)
. It might
be possible that HCC cell lines efficiently converted sulindac to its
sulfide form for effectivity at a relatively lower concentration.

Although the exact mechanism for the antineoplastic effect of NSAIDs
has yet to be delineated, the antiproliferative effect and/or induction
of apoptosis have been attributed to this antineoplastic effect. The
antiproliferative effect of NSAIDs by quiescence of the cell cycle has
been questioned by several authors, but most of them agree regarding
the proapoptotic effect of NSAIDs. In a recent report, Elder et
al.(13)
demonstrated that NS-398, a new generation
of NSAID had no effect on cell cycle distribution in a colon carcinoma
cell line, whereas Piazza et al.(14)
showed
that sulindac produced G1 arrest in proliferating
colon carcinoma cell lines. In this study, we found that both cell
cycle arrest and apoptosis contributed to the growth-inhibitory effect
of sulindac in HCC cell lines. In the preconfluent state, sulindac
produced significant cell cycle arrest in
G0-G1 as early evidence of
an antineoplastic effect; however, this was not time dependent, because
the cells emerged from the arrest and entered into S-phase. In
contrast, the rate of apoptosis increased uniformly in a time-dependent
manner and reached a maximum of 66.5% at 48 h of sulindac
treatment, which was followed by a maximum growth inhibition at 72 h of treatment (Fig. 4)
⇓
.

Several mechanisms have been proposed affecting the molecular pathways
regulating the cellular proliferation and apoptosis by the NSAIDs,
although the key mechanism still remains a dilemma. Only a few of these
mechanisms are related to their capability to inhibit COX, and most of
them are COX independent, including modulation of ras signal
transduction
(15)
, mitogen-activated protein kinase
activation
(16)
, nuclear factor κB activation
(17)
, cyclin expression
(7)
, activation of
the sphingomyelin-ceramide pathway
(18)
, and p53
expression
(14)
. In this study, we used two
well-differentiated HCC cell lines of differentially mutated
p53 and ras genes with an N-ras
mutation at codon 61 position 2 in the HepG2 cell line, and a
p53 point mutation resulted in the amino acid changes of
cysteine for tyrosine at codon 220 in the Huh-7 cell line
(9, 19)
. Sulindac was equally effective in both of these cell lines
and produced similar growth inhibition, indicating that neither of
these pathways is crucial for the antineoplastic effect of sulindac.

HCC is the leading cancer in men in Taiwan and is one of the most
common causes of malignancy-related death in Africa and Asia
(20)
. Despite an enthusiastic effort to diagnose the
disease at an early stage, the prognosis is still dismal, thus implying
that emphasis should be given on preventive measures. Unfortunately, to
date, there is no effective preventive measure in this highly malignant
disease. Both viral infection and chemical carcinogens are thought to
be responsible for the development of HCC, with distinct preceding
steps of premalignant lesions in the vicinity of cirrhotic liver.
Epidemiological studies have shown a 40–50% reduction in mortality
rates from colon cancer in patients receiving NSAID therapy
(21)
. It is worth mentioning that the results of this
study might create a new avenue for the chemoprevention of HCC by the
new generations of NSAID therapy. In the late 1970s, Hial et
al.(22)
demonstrated that COX-dependent NSAIDs
exerted an antiproliferative effect on a rat hepatoma cell line. For
long-term chemoprevention, the COX-dependent NSAIDs are not
suitable because of their deleterious effect on gastrointestinal
functions. Therefore, the new generation of COX-independent NSAIDs such
as exisulind and COX-2-specific inhibitors might be suitable for HCC
chemoprevention. However, before recommending these drugs as routine
prophylaxis, further studies regarding the safety profile of these
drugs and identification of cohorts at high risk for subsequent
development of HCC should be conducted.

Footnotes

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